Hepatitis B virus (HBV) is the infectious agent responsible for several varieties of human liver disease. Many individuals who are infected by HBV suffer through an acute phase of disease, which is followed by recovery. However, a large number of individuals fail to clear their infection, thereby becoming chronic carriers of the infection. HBV infection is endemic to many parts of the world, with a high incidence of infection occurring perinatally from chronically infected mothers to their newborns. The number of chronic carriers worldwide has been estimated at over three hundred million. From this pool of carriers, hundreds of thousands die annually from the long term consequences of chronic hepatitis B (cirrhosis or hepatocellular carcinoma).
The HB virion is composed of two groups of structural proteins, the core proteins and the envelope or surface ("S") proteins. In addition to being the major surface proteins of the virion, i.e., Dane particle, the "S" proteins are the sole constituents of Australia antigen, or 22 nm particles. The "S" proteins are the translational products of a large open reading frame (ORF) encoding 389 amino acids. This ORF is demarcated into three domains, each of which begins with an ATG codon that is capable of functioning as a translational initiation site in vivo. These domains are referred to as preS1 (108 amino acids), preS2 (55 amino acids), and S (226 amino acids) in their respective 5'-3' order in the gene. Thus, these domains define three polypeptides referred to as S or HBsAg [226 amino acids (aa)], preS2+S (281 aa), and preS1+preS2+S (389 aa). Currently available plasma-derived vaccines are composed of proteins containing virtually only the S domain, while yeast derived vaccines successfully developed to date are composed exclusively of the S polypeptide.
The 22 nm particles, or HB surface antigen (HBsAg) particles, have been purified from the plasma of chronic carriers. In terms of their plasma being particle-positive, these chronic carriers are referred to as HBs.sup.+. When these carriers have mounted a sufficient immune response, they can clear the infection and become HBs.sup.-. In terms of their formation of antibodies to HBs, these individuals are denoted anti-HBs.sup.+. In this way, anti-HBs.sup.+ is correlated with recovery from disease. Therefore, the stimulation or formation of anti-HBs.sup.+ by HB vaccines has been expected to confer protection against HBV infection.
This hypothesis has been testable experimentally. Outside of man, chimpanzees are the only species which is fully susceptible to HBV infection, as reflected in quantifiable markers such as HBs.sup.+, and elevated serum levels of liver enzymes. Chimpanzees have been vaccinated with three doses of purified HBsAg particles and then challenged with a large dose of infectious HBV. While mock-vaccinated animals have suffered the signs of acute HBV infection, the HBsAg-vaccinated animals have been protected completely from any signs of infection. Therefore, in this experimental system, HBsAg particles, composed of gp27 and p24 (S domain only), have been sufficient to induce protective immunity. Spurred by these observations, several manufacturers have produced HB vaccines composed of HBsAg particles.
Recently, several independent lines of evidence have suggested the preS sequences may be important in immunity to HBV. The immune elimination of preS antigens during the course of viral infection appears prognostic for viral clearance and abrogation of infection [Budkowska et al., Ann. Inst. Past./Immun. 136D:56-65, (1985)]. During acute hepatitis B, antibodies to preS often arise earlier than antibodies to S [Petit et al., Mol. Immun. 23:511-523, (1986)]. In inbred mice, the immune responses to S and preS appear to be regulated independently, and the presence of preS influences the immune response to S [Milich et al., Proc. Nat. Acad. Sci. USA 82:8168-8172, (1985), J. Immunol. 137:315-322 (1986); Neurath et al., J. Med. Virol. 17:119-125, (1985)]. Furthermore, antibodies to preS neutralize viral infectivity in vitro [(Neurath et al., Vaccine, 4:35-37, 1986)]and preS antigens protect immunized chimpanzees [Itoh et al., Proc. Nat. Acad. Sci. USA 83:9174-9178, (1986)]. In light of these observations and because of the utility of recombinant yeast in producing HB vaccines [Hilleman et al., Vaccine 4:75-76, (1986)], we have formulated experimental preS-containing HB vaccines from recombinant S. cerevisiae.
In order to expand the available supply of HB vaccines, manufacturers have turned to recombinant DNA technology to mediate the expression of "S" proteins. Among microbial systems, Escherichia coli and Saccharomyces cerevisiae have been used most commonly for the expression of many recombinant-derived proteins. Numerous attempts to express immunologically active HBsAg particles in E. coli have been unsuccessful. However, S. cerevisiae has shown great versatility in its ability to express immunologically active HBsAg particles. These particles, when formulated into a vaccine, have proven capable of fully protecting chimpanzees against challenge with live HBV. Furthermore, yeast derived HBsAg has shown the ability to express immuno-logically logically active HBsAg particles which have been as effective in human clinical trials as plasma derived HBsAg [Scolnick et al., JAMA 251: 2812-2815 (1984)]. Therefore, the utility of S. cerevisiae as a host species for directing synthesis of recombinant HBsAg is established firmly. In addition, expression of human therapeutic agents and vaccines in yeast can be very useful for product development, since yeast is free of endotoxin, is nonpathogenic to man, can be fermented to industrial scale, and lacks many of the safety concerns which surround the use of continuous mammalian cell lines (many of which are virally transformed, may be tumorigenic in mice and all of which contain protooncogenes).